Project Summary - Project 1 (Locating General Anesthetic Binding Sites in GABA-A and Glycine Receptors) Our broad goal is to determine the locations in human GABAA receptors (GABAAR) of the binding sites for general anesthetics of diverse chemical structures and the affinities of anesthetics for the different classes of sites. In the previous grant period, we used photoreactive etomidate and barbiturate analogs to establish for the first time that there are at least two classes of intersubunit general anesthetic binding sites in the transmembrane domain of a human GABAAR. One class is located at the interfaces between subunits containing the transmitter binding sites in the extracellular domain and the second class at interfaces not containing the transmitter binding sites. Etomidate binds at anesthetic concentrations with >100-fold selectivity to the first class of sites, and certain barbiturates bind with high selectivity to the second class. We also determined that propofol binds non-selectively to both sites, but only at concentrations greater than necessary to produce anesthesia or potentiate GABA responses, and anesthetic steroids do not bind to either class of sites. Our first aim for this renewal is to use novel photoreactive analogs of anesthetic steroids, barbiturates, and propofol to identify in expressed, human 132 GABAAR additional anesthetic binding sites important for GABAAR potentiation or inhibition. It is our hypothesis that anesthetic steroids bind to intrasubunit sites at the interfaces between transmembrane helices that are also in contact with lipid. A photoreactive analog of a convulsant barbiturate will be used to identify novel binding sites important for GABAAR inhibition. Photoreactive propofol analogs will be used to determine whether propofol binds in a GABAAR to intrasubunit sites equivalent to those identified previously in nicotinic acetylcholine receptors and a prokaryote GABAAR homolog. We will determine in Aims 2 and 3 whether anesthetics bind to equivalent sites in a GABAAR subtype that is expressed extrasynaptically (43d) and in glycine receptors, the receptor most similar in structure to GABAARs and the major inhibitory receptor in the spinal cord. The proposed experiments make use of the Synthetic Chemistry, Protein Chemistry, and Protein Production Cores. Our studies, in conjunction with the development and characterization of novel, potent anesthetics and time-resolved photolabeling studies of Project 2 (Miller) and the functional studies of Project 3 (Forman), should provide an unprecedented view of the number and diversity of general anesthetic binding sites in GABAARs and provide a starting point for the development of anesthetics selective for GABAAR subtypes.